Engineering lanthipeptides by introducing a large variety of RiPP modifications to obtain new-to-nature bioactive peptides

Abstract Natural bioactive peptide discovery is a challenging and time-consuming process. However, advances in synthetic biology are providing promising new avenues in peptide engineering that allow for the design and production of a large variety of new-to-nature peptides with enhanced or new bioactivities, using known peptides as templates. Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs). The modularity of post-translational modification (PTM) enzymes and ribosomal biosynthesis inherent to lanthipeptides enables their engineering and screening in a high-throughput manner. The field of RiPPs research is rapidly evolving, with many novel PTMs and their associated modification enzymes being identified and characterized. The modularity presented by these diverse and promiscuous modification enzymes has made them promising tools for further in vivo engineering of lanthipeptides, allowing for the diversification of their structures and activities. In this review, we explore the diverse modifications occurring in RiPPs and discuss the potential applications and feasibility of combining various modification enzymes for lanthipeptide engineering. We highlight the prospect of lanthipeptide- and RiPP-engineering to produce and screen novel peptides, including mimics of potent non-ribosomally produced antimicrobial peptides (NRPs) such as daptomycin, vancomycin, and teixobactin, which offer high therapeutic potential.


Introduction
Lanthipe ptides re present a major group of ribosomally encoded and post-tr anslationall y modified peptides (RiPPs), pr oduced by a large variety of microorganisms (Arnison et al. 2013 a;Montalban et al.2021 )), including various strains of lactic acid bacteria (LAB), such as Lactococcus lactis , which produces the paradigm lantibiotic nisin ( Fig. 1 A) (Rogers and Whittier 1928, Gross and Morell 1971, Kuipers et al. 1993, 1995Lubelski et al. 2008, de Arauz et al. 2009, Cooper et al. 2010. Lanthipeptides are synthesized from a genetically encoded precursor peptide, LanA, which consists of an N-terminal leader region involved in the recognition of the biosynthetic machinery and a C-terminal core region that under goes post-tr anslational modifications (PTMs). Lanthionine (Lan) and/or methyllanthionine (MeLan) rings are the most c har acterized PTMs in lanthipeptides, whic h ar e intr oduced in a two-step process. In the first step, Ser and Thr residues in the core peptide of LanA are dehydrated to dehydroalanine (Dha) and dehydr obutyrine (Dhb) r esidues, r espectiv el y, by a dehydr atase. Lan/MeLans ar e subsequentl y formed via Mic hael-type addition of sulphydryl groups of Cys residues onto the C β−atom of dehydroamino acids . T he resulting cyclic peptides ha v e constr ained conformations that confer stability and assist in various biological acti vities. Lanthipe ptides can be di vided into five classes based on the biosynthetic enzymes that produce them; the synthetases of these classes have been discussed in pr e vious studies , Repka et al. 2017, Kloosterman et al. 2020, Pei et al. 2022.
In recent years, the rapidly expanding field of RiPPs investigations has r e v ealed a v ast r ange of PTMs that confer important biological properties to these molecules. As of 2021, 41 known classes of RiPPs have been defined, each class defined by its corresponding PTM (Montalbán-López et al. 2021 ). The wide range of moieties installed in RiPPs, such as thiazole/oxazole heterocycles and e po xide groups , ha ve not been reported to occur in lanthipeptides, which suggests that a vast source of PTM enzymes can offer their untapped potential for lanthipeptide engineering to yield nov el exotic structur es in (lanthi) peptides . T hese extensive enzymatic PTMs confer important biological properties to RiPPs, for instance head-to-tail cyclization and the signature cyclic cysteine knot (CCK) motif, which is essential for the anti viral acti vity of cyclotides (Daly et al. 2004, Fu et al. 2021 ). In addition, a N ,Ndimethyl-alanine introduced by the SAM-dependent methyltransferase in the linaridin cypemycin is vital for its antibiotic activity (Claesen and Bibb 2010 ), and a Tyr-Ile ether crosslink provides important contacts for tubulin-binding and thereby potent antimitotic activity of phomopsins (Morisaki et al. 1998, Cormier et al. 2008. RiPP biosynthetic enzymes thus offer a valuable biocatalytic toolbox to install diverse chemical structures into peptides, ther eby conferring nov el bioactivities. Combinatorial a pplication of these tools in lanthipeptide biosynthetic assembly lines would allow for the generation of new-to-nature.
Non-ribosomal peptides (NRPs) (e.g. the antibiotics daptom ycin, vancom ycin, and teixobactin) r epr esent another class of peptide secondary metabolites ( Fig. 1 B), with enormous structural and functional diversity as well as high ther a peutic potential, which has triggered interest in their potential engineering to dev elop impr ov ed antibiotic v ariants (Felna gle et al. 2008, Bozhüyük et al. 2018Liu et al. 2019, Huang et al. 2021, Wenski et al. 2022. Ho w e v er, unlike the biosynthetic plasticity and adaptability of genetically encoded peptides (Montalbán-López et al. 2017 , Wu andvan der Donk 2021 ), NRPs are synthesized by large biosynthetic complexes that are difficult to functionally express and engineer (Felnagle et al. 2008, Kries 2016, Süssmuth and Mainz 2017. To overcome the challenges of NRPS re-engineering to generate impr ov ed NRP v ariants, a str ategy has been proposed to use lanthipeptides as starting points to synthesize peptides with similar NRP structur al featur es b y emplo ying RiPP biosynthetic pathw a ys . (V an Der V elden et al. 2017, Ruijne and Kuipers 2021. As shown in Fig. 2 , the molecular structure of the antimicrobial NRP brevicidine could be partially mimicked by ribosomal synthesis, introducing a cyclic structure by Melan ring formation by the lanthipeptide synthetase involved in the biosynthesis of nisin, NisBC. The r esulting engineer ed lan-thipeptide displayed a similar antimicrobial activity and mode of action as the wildtype NRP br e vicidine, demonstr ating the feasibility of this strategy . Ho w e v er, the construction of biosynthetic assembly lines for the sustainable production of novel lanthipeptides with diverse structures is limited by a number of non-negligible factors . T he substrate specificity of the enzymes that generate the tailoring PTM is one of them, as exemplified by NisBC and the Lan/MeLan ring-installation enzymes involved in nisin biosynthesis. The dehydration activity of Ser/Thr residues of dehydratase NisB depends in part on the amino acids flanking the dehydratable; Ser/T hr residues , e .g. a negativ el y c har ged Asp r esidue pr eceding a serine disfavours its dehydration by wild-type NisB , whereas the cyclase NisC is not capable of catalysing the formation of exceptionall y lar ge rings in peptide substrates (unpublished result). In view of this, only a limited number of substr ates ar e suitable for NisBC-mediated Lan/MeLan ring installation. The leader-dependent mechanism to direct some lanthipeptide PTM enzymes to C-terminal substrate sequences needs to be taken into account when a ppl ying these enzymes in combinatorial biosynthesis (Plat et al. 2011, Montalbán-López et al. 2017. The leader peptides govern the post-translational tailoring processes on specific substrates, eliminating undesired effects on the modification of other peptides/protein products and k ee ping modified pe ptides inacti v e within the pr oducing cell. It may also pose a barrier to the introduction of multiple additional PTMs into one specific substrate (Lagedroste et al. 2020, Lagedroste et al. 2021. The identification of some leader-independent modification enzymes (such as the reductase LtnJ involved in lacticin 3147 biosynthesis, Cotter et al. 2005 ) and the de v elopment of strategies for a combination of different PTM enzymes in one assembly line, such as the 'hybrid leader' strategy, based on the observations that only a limited region of the leader peptide in certain RiPPs is required for efficient modification of the peptides (Burkhart et al. 2017 ), provide possible solutions to this concern. Thus, this calls for the discovery and implementation of more PTM-installation enzymes to form a candidate pool that can be selected from and combined into biosynthetic assembly lines to generate lanthipeptide libraries. Besides dehydration and Lan/MeLan ring formation, an extended range of further modifications such as D-amino acid incor por ation (Ryan et al. 1999, Cotter et al. 2005, halogenation (Castiglione et al. 2008 , Foulston and, methylation (Grigor e v a et al. 2021 ), hydroxylation (Zimmermann et al. 1993, Huo et al. 2017, and acylation (Ozaki et al. 2017 ) have been observed in lanthipeptides. Although the number of reported PTMs and associated enzymes in lanthipeptides is still limited, an expanded r epertoir e of PTMs can be found in other RiPPs classes that offer an extensi ve ad ditional array of structural moieties and bioactivities.
In this r e vie w, we explor e r ecent insights into PTM biosynthetic enzymes from RiPPs, emphasizing their application as catalytic tools in lanthipeptide engineering. We also discuss the combinatorial biosynthesis strategies for efficient diversification of lanthipeptides, including the possibility of structur all y and functionally mimic NRPs. High-throughput methods for screening bioacti ve pe ptides with desir ed ther a peutic pr operties will also be addressed.

RiPP biosynthetic enzymes with engineering potential
PTMs involve enzyme-mediated addition of a wide range of moieties in RiPPs at their amino acid side c hains, suc h as cyclization, hydroxylation, halogenation, and acylation, and can also occur at peptide linkages or at the N-or C-terminus, as in epimerization, β-amino acid incor por ation, and methylation (for se v er al of these structures , see Table 1 ). T hese PTMs pro vide RiPPs with diverse structur es that r egulate their binding and affinity to biological targets and confer promising scaffolds for pharmaceutical applications. Many PTM-associated enzymes have been identified by genome mining across all kingdoms of life and have been produced and c har acterized using homologous or heterologous expr ession systems, whic h hav e allo w ed for a deep biochemical understanding of their catalysis and thereby paved the way for using RiPP maturases in synthetic biology. The enzymes, especially those with exceptional substrate tolerance, allow for the creation of a v ersatile catal ytic toolbox that can be used for lanthipeptide and other RiPPs engineering, ultimately creating novel peptide drugs with desired properties. In this chapter, we discuss various RiPP enzymes that install a diverse set of PTMs and highlight examples and prospects for their engineering application in lanthipeptides.

Dehydr a tion and cyclization
Dehydration is a frequently observed modification across various RiPP families, such as lanthipeptides, linaridins, c y anobactins, and thiopeptides (Ortega and Van Der Donk 2016, Mo et al. 2017, Ma and Zhang 2020, Vinogradov and Suga 2020. Dehydro amino acids often act as an initiation step for further PTMs, as exemplified by lanthipeptides, where Ser and Thr residues in the core peptide of LanA are dehydrated to Dha and/or Dhb residues by a deh ydratase domain. These deh ydroamino acids can be involved in subsequent cyclization to form Lan/MeLan rings and can also be used as substrates for subsequent hydrogenation to generate D-Ala and D-Abu. In addition, dehydroamino acids themselves also provide functionality to peptides. For example, substituting Dha5 for alanine in the lantibiotic subtilin had no effect on its action a gainst v egetativ e Bacillus cereus T cells but abolished its inhibition of spor e outgr owth Hansen 1992 , 1993 ). In addition, the nisin mutant Dha5Ala was shown to have activity very similar to that of wild-type nisin in inhibiting the growth of L. lactis and Micrococcus luteus, but was significantly less active than nisin as an inhibitor of the outgrowth of spores of Bacillus subtilis (Chan et al. 1996 ). Furthermore, a nisin-inactivating enzyme , i.e . present in se v er al nisin-r esistant str ains, was identified as a Dha reductase, whic h inactiv ates nisin as well as subtilin by reducing a Cterminally located Dha to Ala (Jarvis 1967 , Jarvis andFarr 1971 ). Ov er all, these observ ations suggest the important r ole of dehydr o amino acids in the activity and mechanisms of action of certain lanthipeptides, which also offers the basis for the introduction of other PTMs.  (Huo et al. 2017 ); 9 Ref (Tocchetti et al. 2013, Zheng et al. 2016; 10 Ref (Ozaki et al. 2017 ); 11 Ref (Lee et al. 2020a ) and 12 Ref (McIntosh et al. 2011 ). Cyclization is a widespread type of peptide modification that constrains the conformation of peptides , pro viding proteolytic stability , rigidity , and bioactivity (Funk and Van Der Donk 2017 ). The impact of cyclization on membrane permeability, bioavailability, and pharmacokinetic properties has also been studied (Driggers et al. 2008, Naylor et al. 2017, Price et al. 2017. Cyclic RiPPs are generated by ribosomal synthesis of a linear peptide , i.e . subsequentl y modified with heter ocyclic and/or macr ocyclic structur es by a single enzyme or group of enzymes . To date , v arious enzymatic RiPP macr ocyclization r eactions have been revealed, including head-to-tail ligation, lanthionine ring formation, A viCys/A viMeCys ring formation, radical S-adenosylmethionine (SAM)-dependent enzyme-catalysed ring formation, and other types of ring formation brought about by intr amolecular cr osslinks. Se v er al enzymes that catal yse cyclization have been experimentally characterized and successfully applied as synthetic biology tools in peptide engineering, as will be discussed in this section.
Lanthionine (Lan) and/or methyllanthionine (MeLan) rings are c har acteristic structur al featur es of lanthipeptides . T he formation of Lan/MeLan motifs in class I lanthipeptides is catalysed by two different enzymes, a lanthipeptide dehydratase (LanB) and a lanthipeptide cyclase (LanC), which are exemplified by NisB and NisC from the nisin biosynthetic gene cluster. NisBC enzymes are well established for their use in lanthipeptide engineering, as has been shown in many examples . T his includes the installation of a lanthionine ring in v asopr essin (a neur oh ypoph ysial hormone) (Li et al. 2019 ), as well as the production and modification of many ne w-to-natur e lanthipeptides with potent antimicrobial activity (Van Heel et al. 2016 ). For Class II lanthipeptides, a bifunctional lanthipeptide synthetase, LanM, catalyses both dehydration and cyclization of precursor peptides (Table 1 ). SyncM from the marine c y anobacteria Synec hococcus MITS9509 is the most pr omiscuous lanthipeptide synthetase described to date . T he very relaxed substrate specificity of SyncM to w ar ds its precursors and the ability to catalyse the formation of exceptionally large rings in a variety of topologies suggest that SyncM could be an attractive tool to design and produce a variety of ne w-to-natur e lanthipeptides with a broad range of ring topologies (Arias-Orozco et al. 2021. Besides Lan/MeLan rings, the lysinoalanine (Lal) crosslink forms another macrocyclic structure , e .g. found in the lanthipeptide duramycin. The DurN protein from the duramycin BGC is proposed to generate LaI formation by catalysing the addition of the C-terminal Lys to a Dha residue in the peptide in a substrate-assisted, leader peptide-independent way  ).

The
C-terminal 2-aminovin yl-cysteine/2-aminovin yl-3methylcysteine (A viCys/A viMeCys) macr ocyclizations ar e unique structures found in many lanthipeptides (e.g. in epidermin and mersacidin) and also exist in other RiPPs families, including linaridins (e.g. cypemycin), lipolanthines (e.g. microvionine), and thioamitides (e .g. thio viridamide) (Ha yaka wa et al. 2006, Claesen and Bibb 2010, Mo et al. 2019 ). The current model for the biosynthesis of A viCys/A viMeCys involv es a m ulti-step enzymatic process that requires the co-mediation of a dehydratase , cyclase , and flavin-dependent Cys decarboxylase (generically termed LanD in lanthipeptide biosynthesis). The decarboxylase LanD is well c har acterized in se v er al examples, including MrsD fr om mersacidin biosynthesis and CypD from cypemycin biosynthesis (Ding et al. 2018, Mo et al. 2019, Viel et al. 2021. Mor eov er, r ecent investigation of the enzymes involved in AviCys formation, such as MicKC-MicD in microvionin biosynthesis and LxmKXYD for lexapeptide, led to the discovery of the multi-enzyme cooperative biosynthetic strategy for this type of macrocyclization (Wiebach et al. 2018, Lu et al. 2021b. Examples of head-to-tail cyclized RiPPs include large antimicr obial peptides ( e.g. enter ocin AS-48, cir cularin A), c y anobactins from c y anobacteria ( e.g. patellamides and trunkamides), plantderived cyclotides (e.g. kalata B1), orbitides (e.g. cyclolinopeptide A), and fungal peptides (e.g. omphalotin A, α-amanitin and phalloidin) (Mayer et al. 1997, Bell et al. 2000, Kawai et al. 2005, Burgos et al. 2014, Weidmann and Craik 2016. The c y anobactin macroc ylase PatGmac is a subtilisin-like protease involved in head-to-tail cyclization of patellamide. It recognizes the C-terminal macrocyclization signature (positions P1 -P4 ) of AYDG and requires a pr oline r esidue at the P1 position befor e the cleav a ge site. In unmodified peptides, the C-terminal macrocyclization signature is cleaved off to form an acyl-enzyme intermediate, i.e. subsequentl y conv erted to a peptide bond (Koehnke et al. 2012 ). Pat-Gmac allows se v er al c hanges in the substrate sequence, and this substr ate toler ance has been successfull y used to pr oduce a br oad r ange of macr ocyclic peptides in vitro (Houssen et al. 2014 ). Another macrocyclase , OscGmac , encoded within the c y anobactin oscillacyclamide A and B gene cluster, was reported to have an e v en higher substrate tolerance than PatGmac and can process substrates without the conserved proline/thiazoline at position P1. Furthermore, OscGmac can cyclize peptides that are longer than P atGmac substr ates, including peptides containing D-amino acids (Alexandru-Crivac et al. 2017 ). OscGmac thus offers a useful biotechnological tool in peptide engineering to install head-to-tail macrocyclization in a variety of peptides.
Unlike head-to-tail cyclizations formed by attack of the Nterminal amine of the core peptide onto the C-terminus, sidec hain macr olactam/macr olactone ar e cyclizations formed between the N-terminal amine of the core peptide and a side-chain carboxylate or between a side-chain amine and a carbon yl, whic h often occur in the biosynthesis of lasso peptides (e.g. microcin J25 and fusilassin) (Rosengren et al. 2003, Dicaprio et al. 2019 ) and micro viridins (e .g. micro viridin B) (Amaral et al. 2021 ). A recent in vitro study sho w ed that FusC (an ATP-dependent lasso cyclase i.e. homologous to aspar a gine synthetase) fr om the fusilassin pathway is capable of producing millions of sequence-diverse lasso peptides and displays a large substrate tolerance, showing great potential for synthetic biology applications (Si et al. 2021 ).
A subfamily of RiPPs containing distinct intramolecular ω-ester or ω-amide bonds that connect the carboxyl side chain of glutamate or aspartate with a hydroxyl side chain of threonine or serine or with an amine side chain of lysine, named ω-estercontaining peptides (OEPs). The first identified macro-cyclized and well-c har acterized OEPs ar e the micr oviridins (Ishitsuka et al. 1990 ). Recentl y, nov el structur es wer e added to this sub-family, like plesiocin, in which the ester bond is formed by linking of the side chain of amino acids threonine and glutamic acid, catalysed by PsnB (Table 1 ) (Molohon et al. 2011, Lee et al. 2020a ). Thuringinin (Roh et al. 2019 ) and fuscimiditide (Elashal et al. 2022 ) also belong to this sub-family. Genome mining approaches also demonstrated the natural diversity of OEPs (Lee et al. 2020b. Compared with other end-to-side linkage peptides , OEPs ha v e the ob vious c har acteristic of side-to-side connections, whic h ar e modified by ATP-gr asp enzymes, showing a gr eat potential for introducing considerable structural diversification into small peptides . T her efor e, further studies of ATP-grasp enzymes are of great significance as tools for the structural diversification of peptides in synthetic biology. Furthermore, a wide range of macrocyclizations in RiPPs are mediated by radical S-adenosylmethionine (rSAM) enzymes through the construction of C-C and C-S linkages (Mahanta et al. 2017, Benjdia and Berteau 2021, Lu et al. 2021a ). The C-C cr osslinks between ar omatic and aliphatic side c hains of nearby residues occur, e.g. in streptide (Lys-to-Trp) (Schramma et al. 2015 ), pyrroloquinoline quinone (Glu-to-Tyr) (Barr et al. 2016 ), ryptides (Arg-to-Tyr) (Caruso et al. 2019 ), and triceptides (Trp/Phe-to-Asn/Lys/Gln/Ar g/Asp/Ser cr osslinks) (Nguyen et al. 2020 ). The C-S cr osslinks ar e widespr ead in sactipeptides, with subtilosin A as a r epr esentativ e peptide, whic h contains two Cys-Phe crosslinks and one Cys-Thr (S-αC) crosslink (Kawulka et al. 2004, Flühe et al. 2012. In recent bioinformatic research, more RiPPs with C-S cr osslinks wer e identified, suc h as fr eyr asin, whic h contains six Cys-Glu (S-βC) crosslinks, and thermocellin, which contains a Cys-Thr (S-γ C) crosslink . Notably, rSAM enzymes were also found to be able to install six-membered heterocycles into peptides, such as by forming C-O crosslinks between Thr-Gln in r ota peptides (Clark et al. 2019 ) and an α-thioether C-S bond, joining neighbouring Cys and Arg residues in enteropeptins (Clark et al. 2021 ).
RiPPs modified with thiazole/oxazole heter ocycles deriv ed fr om cysteine , serine , and thr eonine r esidues wer e identified in a number of different families, including linear azol(in)-containing peptides (LAPs) (Sinha Roy et al. 1998 ), cyanobactins (Schmidt et al. 2005 ), thiopeptides (Engelhardt et al. 2010 ), and bottromycins (Huo et al. 2012) (Melby et al. 2011. The heterocycles in natural peptides confer stability and/or electronic distributions to the peptides, thus enabling peptide-protein recognition and DN A/RN Apeptide inter actions, whic h ar e thought to be important for the biological function of the modified peptide (Roy et al. 1999, Mhlongo et al. 2020, Mordhorst et al. 2023. The installation of oxazoles and thiazoles into peptides is catalysed by an enzyme complex in two steps. First, a cyclodehydratase catalyses the heteroc yclization of serine/c ysteine/threonine residues. Second, a flavin monon ucleotide (FMN)-de pendent dehydrogenase facilitates the oxidation of oxazoline/thiazoline to produce an oxazole/thiazole moiety, r espectiv el y (Gao et al. 2018, Ge et al. 2019. Se v er al engineering studies hav e explor ed the biosynthesis potential of these cyclodeh ydratase-deh ydrogenase pairs for the generation of oxazole-/thiazole-containing peptide analogues. For example, a hybrid leader strategy was used to direct the cyclodehydr atase LynD (fr om aestur amide biosynthesis) and the dehydr ogenase TbtE (fr om thiom ur acin biosynthesis) to install thiazol(in)es within non-nativ e substr ates (Donia et al. 2008, Hudson et al. 2015, Fleming et al. 2019 ). Furthermore, a leader-peptidefr ee str ategy can catal yse the cyclodehydr ation of leaderless peptide substrates containing a C-terminal Ala-Tyr-Asp recognition sequence by fusing the leader peptide to the N-terminus of the cyclodehydratase (e.g. LynD Fusion and MicD Fusion) (Oman et al. 2012, Koehnke et al. 2015, Oueis et al. 2015, Ge et al. 2019. In addition, dehydr ogenases hav e also been r e ported to acce pt leaderless substrates (Gao et al. 2018 ). These findings led to the fusion of the cyclodehydratase LynD and dehydr ogenase ArtGox, whic h ar e deriv ed fr om differ ent biosynthesis pathwa ys , which allo w ed for the effective installation of thiazole and thiazoline heterocyclic backbones within folded proteins and diverse heteropolymers (Walker et al. 2022 ). This highlights the potential of these enzymes to engineer peptides with thiazole/oxazole heterocycles.

D-amino acid incorpor a tion
D-amino acids ar e non-pr oteinogenic r esidues that ar e pr esent in man y bioactiv e natur al peptides, wher e they enhance the bioactivity and stability of bioactive natural peptides as well as confer ster eoc hemical constr aints for downstr eam biosynthesis into the final products (Ding et al. 2020, Mordhorst et al. 2023. While the occurrence of D-amino acids in RiPPs is r elativ el y r ar e compar ed to their frequent presence in NRPs, a few mechanisms of D-amino acid biosynthesis in RiPP pathways have been elucidated. A small subclass of lanthipeptides contains D-amino acids that are formed by hydrogenation of Dha and/or Dhb residues . T he resulting D-Ala and D-Abu residues have been found in several lanthipeptides, such as lacticin 3147, carnol ysin, and bicer eucin (Ryan et al. 1999, Cotter et al. 2005, Lohans et al. 2014, Huo and Van Der Donk 2016. The enzymes that carry out the reduction reactions are encoded in these lanthipeptide gene clusters, collectiv el y termed LanJ. These enzymes ar e gr ouped into three dehydrogenase classes (Cotter et al. 2005, Repka et al. 2017, including the zinc and NADPH-dependent dehydrogenases (e .g. LtnJ in volved in lacticin 3147 biosynthesis) termed LanJ A , the flavin-dependent oxidoreductases (e .g. CrnJ in volved in carnolysin biosynthesis) termed LanJ B (Lohans et al. 2014 ), and the most r ecentl y discovered F 420 H 2 -dependent reductases (e.g. LxmJ involved in lexapeptide biosynthesis) termed LanJ C .
The engineering potential of LanJ in peptide modification, especially in lanthipeptides, has been explored in se v er al studies. For example, NpnJ A has been shown to r educe dehydr oalanine to D-Ala at non-native positions in a range of non-native substrates, showing flexibility for recognition with respect to the position of the dehydroalanine and high substrate tolerance in both in vivo and in vitro reconstitutions (Yang and Van Der Donk 2015 ). LtnJ A , a r eductase r esponsible for the intr oduction of D-Ala in lacticin 3147, has been co-expressed with the nisin modification machinery (NisBTC) in L. lactis to ac hie v e successful incor por ation of D-Ala into the lanthipeptide nisin and a linear nisin v ariant. Notabl y, LtnJ A does not r equir e a leader peptide for its activity (Mu et al. 2015 ).
In addition, radical S-adenosylmethionine epimerases belong to an enzyme family that catalyses the regiospecific and irr e v ersible intr oduction of m ultiple D-r esidues into ribosomal peptides. Two types of epimerases of this family have been reported in the liter atur e to date (Morinaka et al. 2017 ). PoyD, the first type of r adical SAM epimer ases, installs se v er al D-amino acids along the polytheonamide backbone as a maturation step (T able 1 ). PoyD , as well as se v er al other pr oteusin r adical SAM epimerases (e.g. OspD , AvpD , and PlpD), have been demonstrated to accept a wide range of different substrates and residue types and can introduce different epimerization patterns beyond those observ ed on nativ e substr ates, suggesting that these epimer ases hav e consider able potential for peptide engineering (Morinaka et al. 2014, Vagstad et al. 2019, Korneli et al. 2021. Rational introduction of D-amino acids at desired locations is, ho w e v er, c hallenging as the rules that govern core peptide recognition have not been elucidated to date (Morinaka et al. 2017, Korneli et al. 2021. The second type of radical SAM epimerases is r epr esented by the epimer ase YydG, whic h has a clearl y distinct domain arc hitectur e compared to the PoyD-type of epimerases. YydG installs D-allo-Ile and D-Val into its cognate peptide substrate, YygF. YydF belongs to a novel RiPPs family named e pipe ptides, which was first characterized in B . subtilis . T he bioactivity of epipeptides was demonstrated to be dependent only on epimerizations and requiring no other PTMs (Benjdia et al. 2017 ).
Furthermor e, post-tr anslational epimerizations hav e been r eported in other RiPPs families as well. A r ecentl y discov er ed nov el epimerase, MslH, installs the C-terminal D-Tyr into the lasso peptide MS-271 in a metal-and cofactor-independent manner and exhibits br oad substr ate specificity to w ar ds the N -terminal region of the core peptide (Feng et al. 2018 ). Grisemycin and some other type-A linaridins, such as the salinipeptins and cypemycin, contain multiple D-amino acids in their pr oducts. A r ecent study r evealed that grmL from the grisemycin biosynthetic gene cluster with unknown function is indispensable for grisemycin production, potentially encoding a novel peptide epimerase (Shang et al. 2019, Xiao et al. 2022 ). In addition, the D-Asp in bottromycin A2 is formed in a non-enzymatic epimerization process following the formation of a thiazoline adjacent to an Asp in the precursor peptide . T his spontaneous con version is consistent with pr e vious r eports of epimerization of amino acids adjacent to carboxylated thiazolines Thomas 2013 , Crone et al. 2016 ). Genome mining has r e v ealed the diversity and ubiquity of the family of RiPP epimer ases, pr oviding a broad tool library for introducing Damino acids into peptides, which may facilitate the engineering of lanthipeptides with impr ov ed pharmaceutical properties.

Methylation
Besides proteins, DNA, and RNA, RiPPs also serve as substrates for methylation. Post-translational methylation of peptides is a widespread modification in biological systems, mediated by methyltr ansfer ases that catal yse the addition of methyl gr oups, primarily donated by S-adenosylmethionine (SAM). Peptides are methylated at a number of different sites, including, but not limited to, nitrogen-containing side chains of arginine and lysine, as well as the N-terminus and C-terminus of peptides. Linaridins are a small group within the RiPPs family. All characterized linaridins harbour a conserved α-N -dimethylation through the action of a locally encoded methyltransferase (Mo et al. 2017, Georgiou et al. 2020 ). Cypemycin, a typical linaridin, has a N ,Ndimethyl-alanine introduced by the SAM-dependent methyltransferase CypM (see Table 1 ), which is essential for its bioactivity (Claesen and Bibb 2010 ). Similarly, α-N -dimethylation was also shown to be essential for the antibiotic activity of plantazolicin, a peptide from the thiazole/oxazole-modified micr ocin gr oup of RiPPs, as de-methylated plantazolicin was not active against Bacillus anthracis (Molohon et al. 2011 ). These findings demonstrate the important role of methylation in the biological activity of RiPPs.
Genes encoding methyltr ansfer ases ar e fr equentl y found in Class I lanthipeptide biosynthetic clusters (Acedo et al. 2019, Xue et al. 2022. A r ecentl y c har acterized O-methyltr ansfer ase, OlvS A , which is encoded in a Class I lanthipeptide gene cluster from Streptom yces oliv aceus NRRL B-3009, catal yses the methylation of highl y conserv ed aspartate r esidues to the corr esponding methyl-ester in the cyclic substr ate, whic h is spontaneousl y con verted to succinimide , follo w ed b y non-enzymatic hydr ol ysis to generate a β-amino acid, i.e. isoaspartate in the precursor peptide OlvA. In silico analysis combined with experimental results r e v ealed that OlvS A (termed LanS A ) r epr esents a new family of O -methyltr ansfer ases distinct from protein L -isoaspartate ( Daspartate) O -methyltr ansfer ases (PIMTs). The in vivo and in vitro reconstitution of O -methyltransferase OlvSA activity not only r e v ealed its leader pe ptide-inde pendence and SAM-dependent mechanism, but also laid the groundwork for its potential development as a methyl ester-modification bioengineering tool (Acedo et al. 2019 ). Another SAM-dependent methyltr ansfer ase, annotated as LahS B , was discov er ed within a putative lanthipeptide biosynthetic gene cluster ( lah ), which also contains the unusual Class II lanthipeptide synthetases LahM1/M2. The activity of LahS B was confirmed in vivo (with an E. coli expression system) and in vitro , showing that it methylates the carboxylate of the precursor LahA peptide. Its activity is independent of the leader peptide and shows a certain tolerance to the amino acid r esidues it methylates, potentiall y serving as a methyl esterintroducing tool with high substrate tolerance for lanthipeptide engineering. Although the bioactivity of the peptide LahA is uncertain and the importance of the C-terminal methylation is unclear, modification of the C-terminus of RiPPs is pr obabl y a pr otectiv e str ategy a gainst the action of carbo xype ptidases (Huo et al. 2020 ). The Class II lanthipeptides ar chalan β and ar chalan γ are the first reported lanthipeptides from archaea, both containing one N -terminal methylation, while the associated methyltr ansfer ase genes were identified in their BGCs. Mining of 7157 archaeal genomes revealed chemically diverse and highly v ariable peptide pr oducts, highlighting the potential of archaea as an important source of bioactive peptides as well as novel c hemical structur e-installing enzymes (Liang et al. 2022 ). Besides Classes I and II lanthipeptides, methylation also occurred in the Class III lanthipeptide andalusicin A on its N-terminus. More importantly, α-N dimethylation w as sho wn to be indispensable for its antimicrobial activity (Grigoreva et al. 2021 ). Furthermor e, in r ecent years, a novel class of lanthipeptides, Class V lanthipe ptides, has been re ported, whic h ar e c har acterized by the co-occurrence of lanthionine rings and α-N -dimethylation (Ortiz-López et al. 2020 ). For example, cacaoidin contains an unpr ecedented N,N -dimethyl-lanthionine (NMe 2 -Lan), whic h w as modified b y a putativ e O -methyltr ansfer ase homologue (but displays low sequence similarity to the O -methyltr ansfer ase LanS in Class I lanthipeptide BGCs). Other examples are the lexapeptides, which contain an N,N -dimethyl-phenylalanine installed by the methyltr ansfer ase LxmM, i.e. homologous to the cypemycin α-N -methyltr ansfer ase, and pristinin A3, which contains N,N -dimeth yl-β-meth yllanthionine (NMe 2 -MeLan) introduced by a putati ve carminom ycin 4-O -methyltr ansfer ase (Kloosterman et al. 2020, Ortiz-López et al. 2020. Although the function and enzymology of N -terminal dimethylation remain unclear in Class V lanthipeptides, genome-mining efforts in Class V lanthipeptide BGCs and established heterologous production systems will guide futur e inv estigations on these new lanthipeptide methylations (Román-Hurtado et al. 2021 ). There ar e still man y sur prising discov eries r eported of nov el RiPP methyltr ansfer ases, suc h as the methyltr ansfer ase SinM encoded in the salinipeptin gene cluster, which is predicted to modify N,N -dimethylalanine (Me 2 Ala) in the precursor peptide (Shang et al. 2019 ) , and KgrB, a founding member of a widespread superfamily of Fe-S-containing methyltr ansfer ases, identified in the enteropeptin BGC (Clark et al. 2021 ).
The pr e v alence of methyltr ansfer ases in RiPP BGCs and the importance of methylation on the bioactivity of RiPPs may trigger the de v elopment of methyltr ansfer ases as a useful tool for structure and activity optimization of lanthipeptides (Marsh et al. 2010, Acedo et al. 2019, Grigor e v a et al. 2021, Xue et al. 2022. The bioengineering potential of RiPP methyltransferases has been demonstrated in several studies. For example, cypemycin a-N -methyltr ansfer ase CypM, whic h shows moder ate catal ytic pr omiscuity, can methylate the Class I lanthipeptide nisin and the Class II lanthipeptide halodur acin, wher e methylated nisin shows higher activity than native nisin (Zhang and Van Der Donk 2012 ). The β-methylation, which occurs very rarely in RiPPs, is a common modification in polyketides (PKs) and nonribosomal peptides (NRPs). Characterization of the β-methylation mechanism of RiPP bottromycins revealed the potential to structur all y mimic PKs/NRPs b y emplo ying these novel radical SAM methyltr ansfer ases involv ed in bottr omycin biosynthesis (Gomez-Escribano et al. 2012, Huo et al. 2012, Crone et al. 2016, Franz et al. 2021. The radical SAM methyltransferase domain in OphA (involved in Omphalotin A biosynthesis) showed the possibility to introduce N-methyl groups in non-native substrates, including the NRPS-derived cyclosporin A-like peptides (Van Der Velden et al. 2017 ). Taking all these findings into account, together with the fact that many methylated peptides in natural bioactive peptides exhibit drug-like properties, methylation is a promising tool in lanthipeptide engineering to optimize the activity , stability , and bioavailability of peptide drugs.

Halogenation
Halogenation is catalysed via flavin adenine dinucleotide (FADH 2 )-de pendent or non-heme-iron-de pendent halogenases. These enzymes install halogen atoms (c hlorine, br omine, iodine, or fluorine) into aromatic and aliphatic substr ates activ ated for electrophilic attack, offering more substrate selectivity than haloper oxidases. Halogenated natur al pr oducts hav e been described to occur both in NRPs and RiPPs. Halogens play a vital role in determining the biological activity of these secondary metabolites, as the r emov al or replacement of halogen from the NRP-antibiotics c hlor amphenicol and v ancomycin, e.g. pr ofoundly affected their activity and potency (Harris et al. 1985, Dinos et al. 2016, Wenski et al. 2022. FADH2-dependent halogenases in RiPPs have been shown to catal yse tryptophan-5-c hlorination during the biosynthesis of the lanthipeptide NAI-107 (Castiglione et al. 2008 , Foulston and. Unlike most FADH2-dependent tryptophan halogenases that halogenate free tryptophan, the MibH halogenase involved in NAI-107 biosynthesis acts exclusiv el y on tryptophan in its cognate peptide substrate (see Table 1 ). In addition, MibH r equir es prior modifications to be installed on the NAI-107 peptide for halogenation of the tryptophan indole, and its high substrate specificity limits the potential of using MibH as a general peptide chlorinase (Ortega et al. 2017 ). Inter estingl y, another study r eported that the addition of potassium bromide (KBr) to the growth medium of the pr oducer str ains r eadil y r esulted in the formation of brominated variants of NAI-107. Br can substitute for Cl in microbial metabolites, and this may relate to the higher reactivity of Br − ions compared to Cl − ions and is consistent with the mechanism used by halogenating enzymes . T his result suggests that the Trp halogenases, suc h as MibH, ar e also ca pable of efficientl y incorporating Br into peptides (Cruz et al. 2015 ). Recent work included the RiPP halogenase MibH as a query sequence to mine the marine sponge metagenome for halogenating enzymes, and thus a MibH homologue, named SrpI, was detected in the sponge-derived RiPP/proteusin ( srp ) gene cluster. SrpI catalyses tryptophan-6bromination in the core pe ptide, and, unlik e MibH, SrpI has a certain substr ate toler ance and can br ominate unmodified peptides, thus having good potential as a broad-spectrum peptide tryptophan yl br ominase in biocatal ytic a pplications (Nguyen et al. 2021 ). RiPP halogenases can ac hie v e selectivities that ar e often c hallenging to accomplish using synthetic methodologies, although their substrate specificity limits the development of these enzymes as a gener al-pur pose peptide modification biocatalyst in synthetic biology. Further genome mining and engineering efforts may drive the discovery of those RiPPs halogenases that are better suited for combinatorial applications on a broader series of substrates, opening up the opportunity for functional engineering of novel lanthipeptides (Neumann et al. 2008, Cro w e et al. 2021 ).

β-amino acid incorpor a tion
β-amino acids are amino acids that have a one-carbon extension on the standard α-amino acid backbone. β-amino acid incor por ation confers stability to the secondary structures of peptides and often impr ov es their biological activity (Cabrele et al. 2014, Lee et al. 2017, Evans et al. 2020. Unlike NRPs that contain a wide range of β-amino acids, to date, RiPPs discov er ed containing β-amino acid residues are extremely rare. Ho w ever, recent genome-guided discoveries indicate that ribosomal β-amino acid pr oducts ar e br oadl y distributed and can be biosynthesized within RiPP pathways (Scott et al. 2022, Wang et al. 2022 ). This can be exemplified by the discovery of the PlpX splicease, which is a radical SAM enzyme that functions together with its partner protein PlpY, catalysing an unusual splicing reaction that involv es tyr amine excision fr om the bac kbone and r econnection of the remaining atoms to generate an α-keto-β-amino residue, creating a β-amino acid-containing RiPP metabolite (Morinaka et al. 2018 ) (Table 1 ). Mutational analysis demonstrated the 'XYG' motif present at the splice site, which can direct several splicing e v ents in one precursor. T hus , the PlpXY-mediated reaction can be used for site-specific introduction of multiple α-keto-β-amino acids into gene-encoded precursor peptides (Morinaka et al. 2018 ). Their application was extended to achieve incorporation of various α-keto-β-amino acid residues at either the C -or N -terminal or internal positions of proteins, highlighting their diverse applications in synthetic biology (Lakis et al. 2022 ). Subsequent bioinformatic analysis established that this splicing transformation br oadl y occurs acr oss the bacterial kingdom of life . T he β-amino acid-containing products include many previously unrecognized RiPPs, and β-residues confer potent protease inhibitory activities and expanded structural diversities to these peptides (Scott et al. 2022 ). Another example of β-amino acid installation was found in the Class I lanthipeptide OlvA, which we also mentioned pr e viously to be involved in methylation. The radical SAM-dependent O -methyltr ansfer ase, OlvSA, catal yses the r e-arr angement of a highl y conserv ed aspartate to a β-amino acid, i.e. isoaspartate (Acedo et al. 2019 ). A r ecent study r eported a ne w RiPP, named kintamdin, containing a r ar e β-enamino acid that does not fall into any of the known groups of RiPPs . T he phosphotransferase KinD and the lyase KinC encoded in the kin gene cluster from Streptomyces sp. RK44 are processive in the phosphorylation and elimination of Ser-7 to a β-enamino acid, i.e. (Z) 3-amino-acrylic acid (Aaa), although the underl ying mec hanism of KinCD has not been determined yet (Wang et al. 2022 ). The RiPP-based β-amino acid installation synthases provide an enzymatic route to engineering various β-residues into peptides and proteins, allowing an expanded structural scope and improved stability of lanthipeptides or other RiPPs.

Ornithine incorpor a tion
Ornithine is a non-canonical amino acid, i.e. it is not genetically encoded but is produced in nature via deguanidination of arginine , which pla ys an important r ole in the ur ea cycle. Ornithine is common in many bioactive non-ribosomal peptides, such as the antibiotics daptomycin and gramicidin S, but is rarely found to be incor por ated into ribosomal peptides. Ho w e v er, the r ecentl y identified OspR, a c y anobacterial arginase-like enzyme encoded in the silent osp gene cluster from the c y anobacterium Kamptonema sp. PCC 6506, can install two ornithines in the antiviral RiPP landornamide A (Bösch et al. 2020 ) (Table 1 ). Using OspR as the query sequence, more bacterial arginase homologues were identified fr om div erse RiPP families . T he ar ginase activity of se v er al r epr esentativ es (KspR, PhaR, ChdR, CwbR, BlhR, PacR, and DeaR) was verified in E. coli , and a broad range of peptide sequences were used as substrates to assess their le v el of promiscuity. The results sho w ed that ornithines could be introduced into a wide r ange of substr ates , including some NRP mimics , indicating that these arginases are generally promiscuous . T he high substrate tolerance of these maturases opens up the opportunity for the incor por ation of ornithine by peptide bioengineering . Further study of these ornithine-containing RiPPs and their gene clusters r e v ealed a ne w class of RiPP-deriv ed fattyacylated lipopeptides, the selidamides (Hubrich et al. 2022 ). This new class is characterized by fatty acyl units attached to (hydroxy) ornithine or lysine side chains, which are catalysed by the modifying enzymes of the GCN5-related N -acetyltransferase (GNAT). Taking the biosynthesis of phaeornamide from Pseudophaeobacter arcticus DSM 23566 as an example, the precursor peptide PhaA is firstly modified by PhaR, whic h conv erts ar ginine to ornithine, follo w ed b y hydr oxylation of the ornithine r esidue by PhaI, gener ating 4(S)-hydr oxy-2(S)-ornithine, and subsequent acylation of (hydroxy) for fatty acid attachment, catalysed by the GNAT PhaN. These PhaRIN co-mediated modifications highlight the potential of engineering peptides with diverse, non-ribosomal-like features in a ribosomal way (Hubrich et al. 2022 ). Furthermore, a bioinformatics study discov er ed an unusual type of sactipeptide, termed enteropeptins, featuring N -methylornithine modification. An arginine residue is deguanidinated to ornithine by a predicted Mndependent arginase and then N -methylated by a Fe-S-dependent methyltr ansfer ase, r esulting in the first reported instance of Nmethylornithine in a RiPP (Clark et al. 2021 ). T he disco very of ornithine-installation enzymes and other RiPPs enzymes that catal yse downstr eam modifications based on ornithine enric hes the biocatalytic toolbox and permits an important expansion of the chemical diversity of peptides by bioengineering.

Gl ycosyla tion
Gl ycosylation r efers to the pr ocess of tr ansferring single or m ultiple types of sugar donors to acceptor molecules to form glycosides, the products of which are called glycocins in the RiPPs famil y. Gl ycosylation gener all y occurs on the side c hains of Cys, Thr, or Ser amino acid residues to form S-glycocins or O-glycocins in RiPPs. Compared with other PTM reactions, such as cyclization and lipidation, glycosylation is relatively rare in RiPPs. Until now, only a few glycocins have been identified and characterized. Se v er al studies hav e shown that S-linked gl ycocins ar e more stable than O-linked glycocins, both chemically and biologically (Oman et al. 2011, Wang et al. 2014 ). There are a few S-glycosides formed with UDP-glucose as the natural sugar donor. Sublancin 168, produced by B. subtilis 168, is the first reported Slinked glycocin containing a β-linked glucose moiety attached to the thiol of Cys22 (Oman et al. 2011, Wang and Van Der Donk 2011, Biswas et al. 2017, Ren et al. 2018. The peptide pallidocin also includes a glucose on Cys22, which is catalysed by PalS (Table 1 ), i.e. encoded by an essential gene in the pallidocin biosynthetic gene cluster from the thermophilic Aeribacillus pallidus eight strain (Kaunietis et al. 2019 ). Another S-glycosyltransferase , T huS, is able to catalyse both S-glycosylation of the thiol of cysteine and O-glycosylation of the hydroxyl group of serine in Thurandacin B from Bacillus thuringiensis serovar andalousiensis BGSC 4AW1 (Wang et al. 2014 ). Although the specificities of these enzymes vary, they all have a high substrate tolerance. SunS and PalS can modify various analogues of natural substrates, and SunS is highly promiscuous with respect to its nucleotide-sugar donor and can accommodate different sugars such as UDP-α-D-N-acetylglucosamine (UDP-GlcNAc), UDP-α-D-galactose (UDP-Gal), guanosine diphosphate α-D-mannose (GDP-Man), and UDP-α-D-xylose (UDP-Xyl) to complete the glycosylation reaction (Oman et al. 2011 ). In addition, ThuS demonstrates a high tolerance with respect to both nucleotide sugars and peptide substrates . T huS was able to glucosylate SunA despite the significant differences in the sequences of SunA and ThuA. Ho w e v er, SunS cannot modify ThuA, indicating that the tolerance of ThuS to peptides is higher than that of SunS (Wang et al. 2014 ). Structural and mechanistic investigations of ThuS and SunS r e v eal that they consist of an unusual gl ycosyltr ansfer ase Type A (GTA)-fold arc hitectur e and form a dimer to create an extended cavity to accommodate different peptide substrates (Fujinami et al. 2021 ). Ov er all, these Sgl ycosyltr ansfer ases offer promising potential to be used as a tool for the biosynthesis of glycosylated bioactive peptides. Furthermor e, the S-gl ycoside gl ycocin F (GccF), i.e. secr eted by Lactobacillus plantarum KW30, has an N -acetylhexosamine and not a UDPglucose S-linked to Cys43, which is indispensable for its activity (Stepper et al. 2011, Drummond et al. 2021. Finally, the glycocin F homologue ASM1, produced by Lactobacillus plantarum A-1, differs fr om gl ycocin F in the C-terminal sequence of the peptide (Main et al. 2020 ).
In addition to S-gl ycocins, O-gl ycocins that attac h a sugar moiety to Ser or Thr r esidues hav e also been r eported, suc h as Enterocin F4-9 (Maky et al. 2015 ) and Enterocin 96 (Izquierdo et al. 2009 ). Enzymes derived from this family are amenable to peptide engineering as gl ycosyltr ansfer ases hav e a r elativ el y br oad and relaxed donor-and acceptor-substrate scope (Nagar and Rao 2017 ). The sugar donor of O-glycocins can also be attached to Tyr residues, like in the previously mentioned Class V lanthipeptide cacaoidin (Román-Hurtado et al. 2021 ). Furthermore, a glycosylated lanthipeptide, named NAI-112, has also been discovered. It contains a deoxyhexose modification, i.e. N-linked to a tryptophan r esidue, whic h is quite r ar e in the RiPPs family (Sheng et al. 2020 ). Glycosylation also occurs in lasso peptides, although their mechanism of action is unclear to date (Zyubko et al. 2019 ).

Hydr o xylation
Hydroxylation is a relatively abundant modification in both NRPs and RiPPs. Hydroxylation in the RiPPs family can occur either on aromatic moieties or on aliphatic amino acids to produce antibiotics with altered activity.
Thiope ptides re present an important RiPP class equipped with v aried and r emarkable bioactivities (Arnison et al. 2013 ). The fam-ily of thiopeptides typically features a characteristic macrocyclic cor e, whic h is composed of a six-member ed, nitr ogenous thiazole and multiple oxazoles and dehydroamino acids (Arnison et al. 2013 , Zhang and. Some thiopeptides contain hydr oxyl gr oups that ar e intr oduced b y c ytoc hr ome P450 hydr oxylases (Liu and Thomas 2013, Tocchetti et al. 2013, Zheng et al. 2016. Thiopeptide GE2270 contains a hydroxyl group located at the β-position of the amino acid phen ylalanine, whic h is introduced by the cytoc hr ome P450 monooxygenase PtbO, i.e. encoded in the gene cluster of the GE2270 producer Planobispora rosea (Tocchetti et al. 2013 ). In addition, the Ile10 of thiostrepton is dihydr oxylated, whic h is catalysed by TsrR, another cytoc hr ome P450 enzyme (Zheng et al. 2016 ). Furthermor e, two differ ent types of hydroxyl groups exist in nosiheptide; one is attached to the γposition of Glu6, which is a typical sp 3 carbon. Another is put at the Pyr3 position, which is a rare sp 2 carbon atom. These two modifications are performed by NosB and NosC, respectively, which belong to the P450 monooxygenases (Liu and Thomas 2013 ).
Hydroxylation is also reported in a family of lasso peptides, whic h featur e a unique lariat-knot arc hitectur e . T he first identified hydroxylated lasso peptide was Canucin A, which was discov er ed by activ ation of its silent biosynthetic gene cluster in Streptomyces canus . Further studies on the biosynthetic pathway of Canucin A sho w ed that CanE, an a-ketoglutar ate/ir on (II)de pendent hydro xylase, could install the hydro xyl group at the β-carbon of the C -terminal aspartate residue on the precursor peptide, which occurs prior to macrocyclization. In addition, CanE could also modify Asn and Glu effectiv el y but not Ala and Ser, which indicates that CanE has a certain substrate specificity, i.e. it is promising as part of a toolkit for the combinatorial biosynthesis of lasso peptides (Zhang and Sey edsay amdost 2020 ).
The β-hydroxylation of Asn can also be found in polytheonamides, whic h ar e among the first known members of the proteusin family of RiPPs . P olytheonamides represent the most heavil y post-tr anslationall y modified biomolecules deriv ed fr om amino acids described to date. Se v en enzymes are responsible for installing ∼50 PTMs distributed across 49 residues, including epimerization, deh ydration, meth ylation, and h ydroxylation, among others. Co-expression studies confirmed that PoyI regioselectiv el y hydr oxylates V al24, V al32, and Asn38. The V31 L and N38H of PoyA point m utants wer e toler ated b y Po yI, while V31H and N38Q were not . The recognition motifs of PoyI need to be further studied to enable the use of these enzymes as hydroxylation tools in synthetic biology.
Modification of Asn by hydroxylation also occurs in lanthipeptides, whic h gener all y contain lanthionine (Lan) and/or methyllanthionine (MeLan) rings. Duramycin consists of 19 amino acids and includes one Lan and two MeLans, an unusual lysinoalanine (Lal) bridge, and an erythr o-3-hydr oxy-L-aspartic acid. The modification of hydroxy-Asp is installed by an α-ketoglutar ate/ir on (II)-de pendent hydro xylase, DurX (Table 1 ). A gl ycine r esidue next to the Asp residue is necessary for DurX activity, which was determined by mutational analysis of the DurA precursor peptide (Zimmermann et al. 1993, Huo et al. 2017. Further studies on these peptides and their deriv ativ es pr ov e that hydroxylation is important for the bioactivity of peptides (Ökesli et al. 2011(Ökesli et al. , Huo et al. 2017. Hydroxylation in other amino acids than Asn can also be found in lanthipeptides . T he di-hydroxylation of a pro residue by a cytoc hr ome P450 enzyme in the biosynthesis of the lanthipeptide NAI-107 has been demonstrated (Maffioli et al. 2014 ).
The hydroxylation modification is also present in the dikaritins, lik e ustilo xin B (Ye et al. 2016 ) and asperipin-2a (Ye et al. 2019 ), Figure 3. Natural combinations of diverse modifications in lanthipe ptides. Cinnam ycin and duramycin contain a hydroxyl group, which is essential for their antimicrobial activity. This group is marked in green in the Cinnamycin chemical structure; Lanthipeptide microbisporicin (NAI-107) contains a 5-c hlor otryptophan (5-Cl-Tr p) motif, whic h is shown in gr een in the corr esponding structur e; NAI-112, a gl ycosylated Class III lanthipeptide, contains a r ar e deoxyhexose modification N-linked to a tryptophan residue . T his structural feature is highlighted in green.
although the mechanism of hydroxylation has not been elucidated to date. Although Asn-hydroxylation enzymes were found in different classes of RiPPs, the enzyme recognition motifs were different. Further studies on specific enzyme motifs are needed to use these enzymes for synthetic biology.

Pren yla tion
Pr en ylation modification can increase the diversity of the (modified) peptide structure by adding carbon chains of different lengths at various positions of the peptide, thereb y endo wing the peptide with increased metabolic stability, enhanced membrane interactions , better peptide bioa vailability, and impro ved pharmacokinetic and pharmacodynamic properties (Zhang and Bulaj 2012 ). Due to the significant impact on the properties of peptides following lipidation, lipidation has pr ov en to be an effective tool in peptide engineering. There are some naturally occurring lipidated peptides with excellent antibacterial activities in NRPs, such as polymyxin B, which is active against Gram-negative bacteria (Velkov et al. 2010 ), and daptomycin, which kills Gram-positive bacteria (Baltz 2009 ). Recently, an increasing number of lipid modifications on peptides have also been found in RiPPs, in which pr en yltr ansfer ases (PTases) fr om c y anobacteria play an important role.
The pr en yltr ansfer ases fr om c y anobacteria (PTases) usually perform forw ar d or r e v erse pr en ylation (5-carbon) or ger an ylation (10-carbon) reactions on Ser , Thr , or Tyr to pr oduce v arious c y anobactins. In-depth studies performing structural, molecular dynamics, and biochemical characterization of prenyltransfer ases fr om c y anobacteria sho w that different enzymes have different specificities for their donors and acceptors. For instance, LynF (Table 1 ), P a gF , PirF , and SphF can all accept tyrosine but recognize different donors (McIntosh et al. 2011, Hao et al. 2016, Martins et al. 2018, Morita et al. 2018. LynF, P a gF, and SphF utilize 5-carbon moieties as donors, while PirF prefers a 10-membered carbon chain for the ger an ylation r eaction. For some enzymes, e v en when recognizing the same donor and acceptor, the orientation of the pr en yl gr oup sometimes also v aries. P a gF and SphF carry out forw ar d pr en ylation, while LynF performs r e v erse pr en ylation. The r e v erse O-pr en ylated Tyr then under goes a spontaneous Claisen r earr angement to yield a forw ar d C-pr en ylated pr oduct. Pr en ylation on Tr p r esidues has also been discov er ed, wher e usuall y pr en ylation is performed at the N -terminus of the pe ptide, lik e for AcyF (Dalponte et al. 2018 ), or at the C-3 carbon of the indole ring, like for KgpF (Ishida et al. 1997 ). In addition to modifying the amino terminus of some linear c y anobactins b y, for instance, AgeMTPT and MusF1/2, N-forw ar d mono-or bis-pr en ylation also occurs on ar ginine, catal ysed by AgcF (Phan et al. 2021 ). Recently, a unique and bifunctional prenyltr ansfer ase, LimF, originating fr om Limnothrix sp . CA CIAM 69d, has been reported that catalyses histidine-C-geranylation and Tyr-O-ger an ylation. Inter estingl y, it can act on various non-natural substrates (Zhang et al. 2022 ). In fact, the prenyltransferases isolated and identified from c y anobacteria display a wide substrate scope. In addition to modifying their own natural cyclic peptide substr ates, LynF, P a gF, and PirF could also modify linear peptides and e v en single amino acids. As these enzymes have been discov er ed r ather r ecentl y, ther e ar e not man y examples of the introduction of pr en ylation into lanthipeptides, but based on the observed relaxed substrate specificity of these enzymes, the engineering of PTases to pr oduce ne w-to-natur e lanthipeptides will be an ongoing and important trend in the near future.
In addition, longer aliphatic chains can be introduced by the pr en yltr ansfer ase ComQ involv ed in the biosynthesis of the Bacillus quorum-sensing pheromone ComX. ComQ introduces geranylation or farnesylation of a 15-membered carbon chain at the C-3 position of a Trp. The ComQ natto from B. subtilis subsp. natto can accept a wide range of N -and/or C -terminally truncated analogues, e v en a single Tr p. This demonstr ates that ComQ natto has a broad substr ate toler ance, whic h opens up the potential of ComQ as a Figure 4. Sc hematic dia gr am of further modifications and combinations intr oduced into a br e vicidine mimic. The implementation of mimic king mainly includes four parts . T he fatty acid chains can be mimicked by using hydrophobic amino acids, or they can be catalysed by enzymes . T he mimic of a lactone ring by a lanthionine and the fatty acid mimicking by three hydrophobic AA residues have already been achieved . Further modification work mainly focuses on the incorporation of D-amino acids, the non-canonical amino acid ornithine, and the mimicking of a fatty acid chain by enzymatic methods. Piel's group has demonstrated the possibility of introducing D-amino acids and ornithines onto similar linear peptide sequences .
new tool for the introduction of long carbon chains in lanthipeptide engineering (Sugita et al. 2018 ).

Acylation
Acylation can introduce an acyl or an acetyl group onto a peptide, which is common in NRPs but not as well represented in RiPPs. At pr esent, onl y a fe w peptides with acetylation modification derived fr om RiPPs hav e been identified, mainl y in lanthipeptides (Ozaki et al. 2017, Wiebach et al. 2018, Hubrich et al. 2022 ) and lasso peptides (Zong et al. 2018 ).
Acylation in RiPPs mainly occurs at the N-terminus of a peptide . In goadsporin, after remo val of the leader peptide, the Nterminal amino group was acetylated in the presence of acetyl-CoA by GodH (Table 1 ), which is a GNAT domain-containing acetyltr ansfer ase (Ozaki et al. 2017 ). Acylation at the N-terminus of the peptide can also be follo w ed b y other biosynthetic pathwa ys , like NRPs or PKS. Microvionin and Nocavionin contain a bis-methylated guanidino fatty acid attached to the acylated Nterminus of the peptide, which is deriv ed fr om fatty acid or polyke-tide biosynthesis (Wiebach et al. 2018(Wiebach et al. , 2020. The Piel group has r ecentl y r eported the selidamides, whic h form a ne w famil y of ribosomall y deriv ed, fatty-acylated lipopeptides . T he fatty acyl moieties are attached to the side chain of lysine or (hydroxy)ornithine . T he acylation reaction was catalysed by maturases of the GCN5-related N-acetyltransferase superfamily (Hubrich et al. 2022 ).
Acylation also occurs natur all y in lasso peptides. Albusnodin is the first confirmed lasso peptide wher e ther e is experimental demonstration of an acetylation modification, which is a pr er equisite for the biosynthesis of this kind of lasso peptide (Zong et al. 2018 ).
For acetylation, compared to using acetyl-CoA as a donor, the enzyme involved in lipolanthine synthesis has obvious advantages. In addition to accepting diverse donors, it can also directly modify peptide side chains rather than being limited to the Nterminus of the peptide, which is of great significance for increasing the diversity of peptides . T his makes it possible to generate mor e activ e peptides with different acyl chain lengths in vivo . At  (Bosma et al. 2011, Urban et al. 2017, Hetrick et al. 2018. (B) Bacterial r e v erse two-hybrid tec hnology for identifying a lanthipeptide inhibitor of the p6-UEV PPI (Yang et al. 2018 ).
the same time, more genetic and biochemical characterization is r equir ed to obtain a more comprehensive and deeper understanding of the related acetyltransferases, which are certainly promising tools for synthetic biology.

Epoxidation
Epoxidation modification involves the conversion of a double bond to a thr ee-member ed ring, whic h is close to an equilateral triangle. Epoxides are usually highly reactive because of their high ring tension and polarized carbon-oxygen bonds (Fretland and Omiecinski 2000, Tang 2007, Amacher 2012, Morisseau 2013. The introduction of an e po xide group into peptides usually makes them easy targets for ring opening by nucleophilic groups such as an amine (-NH 2 ) or thiol moieties (-HS-), which are frequently occurring in peptides (Brotzel and Mayr 2007 ). Since there are man y pr oteins and r eactiv e amine gr oups in biological systems, the new scaffolds typically undergo further enzymatic modification to generate mature and stable peptides . T he cytochromes P450 can act on aromatic or double bonds of peptides to install e po xidation modifications in peptides (Lamb et al. 2013, Rydberg et al. 2014 ).
Epoxide modification occurs in a variety of natural products, including NRPs as well as RiPPs. In NRPs, cyclomarin A and cy-clomarin B are equipped with the structural characteristics of e po xidation. This modification is performed by a cytoc hr ome P450 enzyme named CymV (Schultz et al. 2008 ). Similarly, thr ee-member ed rings ar e also pr esent in the structur al featur es of RiPP-derived peptides . For instance , during the biosynthesis of thiostr epton, the cytoc hr ome P450 enzyme TsrI carries out e po xidation of the quinaldic acid moiety of thiostrepton (Table 1 ). Epoxidation is imper ativ e for subsequent modification steps to pr oduce matur e thiostr epton (Tocc hetti et al. 2013, Zheng et al. 2016.
Due to the extr emel y high tension and reactivity of e po xides, further modifications are generally performed in vivo to produce mor e stable pr oducts. But e v en so, e po xides display important activities, especially anti-cancer activity, whic h deserv es further investigation.

Na tur al combina tions of PTMs in lanthipeptides
In addition to the primary modification that forms the thioether rings, some lanthipeptides also contain various secondary modifications that introduce specific functional groups, and these diverse secondary modifications have a great impact on activity or stability or are necessary for the lanthipeptide synthesis or structur e itself. Examples ar e shown in Fig. 3 , wher e the hydr oxylation of Asp15 of cinnamycin or duramycin is essential for their antimicrobial activity, as the hydroxyl group can form a hydrogen bond with the ammonium group of ethanolamine, which is part of the tar get l ysophosphatidyl-ethanolamine (Hosoda et al. 1996, Ökesli et al. 2011, Huo et al. 2017. Furthermore, NAI-112 is a glycosylated Class III lanthipeptide produced by an Actinoplanes sp. strain with potent bioactivity against nocice pti ve pain, while weak antimicrobial activities are also displayed. It contains two labionin/methyllabionin motifs, and it also contains a r ar e deoxyhexose modification N-linked to a tryptophan r esidue, whic h is catalysed by the tailoring enzyme AplG (Sheng et al. 2020, Tocchetti et al. 2021. Another lanthipeptide, microbisporicin, contains 5-c hlor otryptophan (5-Cl-Tr p), whic h is formed by a halogenase named MibH. The halogenation modification is important for the activity of microbisporicin, because the 5-Cl-Trp residue is dir ectl y next to the presumed binding site of the N-acetylmuramic acid moiety (Cruz et al. 2015, Maffioli et al. 2016, Ortega et al. 2017. The combination of these natural Lan or MeLan with various tailoring modifications in lanthipeptides reflects the possibility of combining different modifications that confer diverse biological activities into one peptide, which is an inspirational motivation for cr eating ne w-to-natur e molecules with specific and desir ed biological activities. In addition, enzymes with a high substrate toler ance, suc h as DurX, present in lanthipeptide biosynthetic path-ways also create unprecedented possibilities for the introduction of div erse 'for eign' modifications into lanthipeptides. As these tailoring modifications also occur in other RiPPs families, it is likely that some of these enzymes ma y ha ve been cross-combined at the genetic le v el. These notions all indicate the feasibility of combinatorial biology r esearc h with lanthipeptides as the starting point for engineering multiple modifications into lanthipeptides.

Artificial combinations of PTMs into lanthipeptides
As RiPPs are synthesized by ribosomes, the primary sequence of the peptide can be changed by site-directed mutagenesis, which r equir es a certain substrate flexibility of the PTM enzymes of RiPPs. Se v er al studies hav e shown the feasibility of this strategy. A large number of mutants of various lantibiotic precursor peptides can be produced by site-directed mutagenesis employing the biosynthetic mechanism in a heterologous or homologous host (Kuipers 1996, Cortés et al. 2009, Cooper et al. 2010. Both lacticin 3147 and mersacidin wer e inv estigated by full alanine scanning (Cotter et al. 2006 ). Random m uta genesis is also an a ppr oac h with a high potential to generate vast genetically encoded libraries of natural-like lanthipeptides containing substantial structural diversity. For example, a precursor gene-encoded library of 10 6 lanthipeptides has been generated in vivo by employing the promiscuous lanthipeptide synthetase Pr ocM, whic h led to the identification of an HIV p6 protein-human TSG101 protein interaction inhibitor library screening (Yang et al. 2018 ). Furthermor e, Sc hmitt et al. used a large library of a variety of ring elements from 11 different lantibiotics to screen for impr ov ed activity with a novel nano-Fleming technology based on micro-alginate beads with a fluorescent producer target cells, and the protease NisP. In this way, they isolated > 20 ne w-to-natur e peptides with impr ov ed bioactivity (Sc hmitt et al. 2019 ). In addition, the in vivo activity of NisC allows for the cyclization of a wide arr ay of unr elated and designed peptides that were fused to the nisin leader peptide (Kluskens et al. 2005, 2007. These in vivo studies have proven the remarkable promiscuous nature of lantibiotic biosynthetic enzymes. Lantibiotic biosynthetic enzymes can also install dehydro-amino acids or thioether rings in a large variety of non-native peptides attached to the native leader peptides. For example, the lanthipeptide synthetase LctM can not only modify lacticin 481 mutants, but is also able to modify other unrelated peptides containing thioether rings (Chatterjee et al. 2006 , Le v engood and v an der Donk 2008 ). These studies highlight the promiscuity of these enzymes, although not all lantibiotic biosynthetic enzymes exhibit natural substrate promiscuity, which imposes limitations on installing lanthionines on nonnatur al substr ates. Ho w e v er, lanthipeptide synthetases can be en-gineered to display different substrate promiscuities. For instance, a dehydratase mutant library of NisB with 10 5 variants was generated via error-prone PCR. Subsequent high-throughput screening (HTS) based on cell surface display of the peptide products r e v ealed a NisB variant that sho w ed substrate flexibility against non-natur al substr ates . Although m uta genesis of core sequences and protein engineering of PTM enzymes can increase the diversity of peptide structures, the structural variety that can be generated by such means is still limited.
As many modification enzymes in RiPPs recognize a leader or part of the leader to modify core pe ptides, di verse modifications fr om differ ent biosynthetic pathways within the same family or acr oss famil y boundaries can be combined into various core peptides by redesigning the leader peptide. In this way, se v er al RiPP mac hineries hav e been combined to generate new and novel natural peptide mimics.
Nisin is one of the most studied lanthipeptides and is widely used as a natural food preservative . T he nisin synthesis machinery was used to modify and secrete a putative two-component lantibiotic of Streptococcus pneumoniae , which was ac hie v ed by genetically fusing the sequences of spr1765 (pneA1) and spr1766 (pneA2) to the nisin leader-encoding sequence . T he resulting RiPP harbours m ultiple dehydr ated serine and/or thr eonine r esidues and (methyl) lanthionines. Both modified peptides displayed antimicrobial activity against Micrococcus flavus (Majchrzykiewicz et al. 2010 ). Chimeric leader peptides enable the combination of modifications introduced by RiPP maturases from unrelated pathwa ys , given that the chimeric leader generated contains the corr esponding r ecognition sequence (RS) for m ultiple modification enzymes. In this way, a chimeric leader containing recognition sequences for NisB/C and the thiazoline-forming cyclodehydratase HcaD/F enabled the creation of a new-to-nature thiazoline-lanthipeptide Class I hybrid. When the recognition sequence of the NisB/C leader sequence w as sw apped with the recognition sequence of ProcM, thiazoline-lanthipeptide Class II hybrid molecules could be gener ated. Furthermor e, a thiazolinesactipeptide hybrid could also be produced by this a ppr oac h (Burkhart et al. 2017 ). T hus , the chimeric leader peptide strategy holds vast potential for combinatorial ne w-to-natur e peptide generation.
Combinatorial RiPP biosynthesis may also lead to the generation of NRP mimics, as m uc h of the chemical complexity present in NRPs is also found in RiPPs. Since NRPs are not directly geneencoded and are synthesized through large, multi-modular enzymes, the generation of NRP variants is challenging. T herefore , in recent years, the possibility of making mimics of NRPs with increased biological activity or improved physical or chemical properties by RiPP biosynthetic pathways has attracted a lot of interest. The Kuipers group initiated an important step to w ar ds this goal. They described a strategy to synthesize NRPs, mimics of the r ecentl y discov er ed non-ribosomal antimicr obial peptide br evicidine, employing nisin biosynthetic enzymes (Fig. 2 ). In this work, the lactone moiety was replaced by a thioether ring, and the fatty acid chains of NRPs were mimicked by a few hydrophobic amino acids . T he engineered mimics sho w ed antimicrobial activity against Xanthomonas campestris , which demonstrated that the structur al mimic king of NRP by RiPP biosynthesis is feasible and offers great opportunities for engineering a wide range of effective antibiotics . In addition, studies by Piel's group have shown that OspR was able to modify three Arg residues to the non-canonical ornithine in another linear br e vicidine mimic. In addition, the epimerase OspD could introduce a D-amino acid in this mimic, although the position of the D-amino acid did not match the D-amino acid positions in natural brevicidine . These results indicate that there are a variety of RiPP tools available that can potentially be combined to introduce NRP structural features into ribosomal peptides (Fig. 4 ).

Screening ne w-to-na ture lanthipeptides with different methods
As illustrated in the preceding sections, large genetically encoded libraries of lanthipeptide variants can be generated by mutagenesis, which calls for the development of novel methods for the identification of desir able v ariants. Gr eat efforts have been made to de v elop HTS methods to select v ariants fr om lar ge libr aries a gainst specific tar gets, whic h can then be pr epar ed in lar ge quantities for further structure and bioactivity characterization.
Surface display technologies, including bacterial, yeast, and phage display technologies, represent an effective means of identifying peptides that bind to a cell, protein, or other molecules of inter est. Differ ent surface display tec hnologies hav e been used in lanthipeptide screening, as shown in Fig. 5 A. The first successful lanthipeptide surface display system was constructed in L. lactis and demonstrated to be effective in screening libraries with up to 10 9 variants of lanthipeptides (Bosma et al. 2011 ). Other examples of successful selection of desirable lanthipeptide variants have been shown for the Class II lanthipeptide lacticin 481, for which an analogue with novel binding activity for αv β3 integrin was scr eened fr om a 6 × 10 5 libr ary b y y east surface display combined with fluor escence activ ated cell sorting (FACS) as potential ligands for tumour ima ging. (Hetric k et al. 2018 ). In the same study, a nisin variant with a different lipid II binding mode than the native nisin was identified from a library encoding 1.2 × 10 6 variants by Nterminal phage display (Hetrick et al. 2018 ). With the knowledge that C-terminal fusions should be adv anta geous for the study of pr otein-pr otein inter actions r equiring fr ee carboxy-termini (Fuh and Sidhu 2000 ), a C-terminal phage display system was developed. Applying this system, artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin wer e r eadil y identified fr om lar ge C-terminal display libr aries (Urban et al. 2017 ). Not limited to lanthipeptides, the bacterial surface display technique was also successfully implemented for the screening of high-affinity ligands specific for the VEGFA binding site on neuropilin-1 from a 6 × 10 9 member bacterial display libr ary deriv ed fr om the cyclotide Kalata B1 scaffold, and a C-to-N c yclized c yclotide variant w as identified as a potent antagonist of neuropilin that inhibits endothelial cell migration (Getz et al. 2013 ).
In addition, a bacterial r e v erse two-hybrid technology was developed that allo w ed for the identification and characterization of pr otein-pr otein inter actions , leading to the disco v ery of antivir al lanthipeptide variants that prevent the interaction between the HIV p6 protein and the UEV domain of the human TSG101 protein, an inter action i.e. r equir ed for the HIV vir al budding pr ocess (Yang et al. 2018 ). One interaction inhibitor was screened out from a 10 6 -non-natural lanthipeptide library, which was constructed in E. coli using the substr ate-toler ant lanthipeptide synthetase ProcM (Fig. 5 B). Suc h libr aries, containing substantial structur al div ersity, may be combined with other cell-based assays to identify lanthipeptides with new biological activities (Yang et al. 2018 ).
Furthermor e, colon y-based assays ar e widel y utilized for HTS of natural product analogues and have been exploited in a recent study to screen Class II lanthipeptide haloduracin analogues by in-colon y r emov al of leader peptides in E. coli (Si et al. 2018 ). The main design principle underlying this approach is cellular compartmentalization, wher e the post-tr anslational modifications of precursor peptides are completed in the cytosol and the leader peptide r emov al is pr ogr ammed by a protease located in the periplasmic space. Subsequentl y, autol ysis of E. coli cells is induced, permitting extr acellular r elease of the final pr oduct for biological activity screening and analysis . T his method is suitable for HTS of RiPPs variants that are inactive in the presence of the attached leader peptide (Si et al. 2018 ).
Mor e r ecentl y, the nano-Fleming, a miniaturized and parallelized high-throughput inhibition assay, was developed to screen 6000 combinatorial lanthipeptide variants at the nanoliter (nL) scale . T he fluor escentl y labelled peptide producer and sensor cells wer e enca psulated into nanoliter r eactors (nLRs) for gr owth and peptide production. The nLRs with a small number of sensor cells were identified as containing potential producers of lanthipeptide variants, as shown in Fig. 6 . With this combinatorial a ppr oac h, a number of antimicrobial lanthipeptides that sho w ed impr ov ed activity over wild-type peptides or were able to bypass resistance mec hanisms wer e identified (Sc hmitt et al. 2019 ).
The studies of high-throughput sequencing methods in other RiPP families also provide concepts that can be extended to lanthipeptides . For example , mRNA displa y technology compatible with Flexizyme r epr ogr amming allows the intr oduction of nonnatural amino acids into the RiPP antibiotic pantocin, with the capacity to screen large and diverse libraries containing multiple sim ultaneous m utations (Fleming et al. 2020 ). Various screening methods that have been developed so far and that are expected to be de v eloped in the futur e continuousl y foster the e volution of lanthipeptide engineering. Ac hie ving the discov ery of ne w-tonature lanthipeptides with enhanced pharmacological properties and ther a peutic possibilities fr om lar ge in vivo lanthipeptide engineering libraries is a challenging but realistic goal.

Conclusion and outlook
The diverse PTM enzymes from RiPPs constitute a valuable biocatalytic toolbox that can be used for lanthipeptide and other RiPP engineering. Combinatorial application of these tools in lanthipeptide biosynthetic assembly lines allows for an expanded structural scope and enhanced or altered bioactivities of lanthipeptides, enabling the de v elopment of an inv aluable r esource for bioactive compounds and potential drugs. Several strategies for the combined use of these enzymatic tools have been proposed and successfull y demonstr ated in the above-mentioned studies. In ad dition, leader-inde pendent enzymes, such as the dehydrogenase LanJ and the O -methyltr ansfer ase OlvsS A , ar e well suited for introducing additional RiPPs modifications into various cor e peptides. Mor eov er, enzymes with high substrate tolerance, such as the lanthipeptide synthetase SyncM, the prenyltr ansfer ase ComQ, and the peptide arginase OspR, are good candidates for combinational use to create non-natural peptides. Ho w e v er, the implementation of some RiPPs enzymes in combinatorial biosynthesis is still challenging; e.g. the halogenase MibH r equir es prior modifications to be installed on the precursor peptide for halogenation of the tryptophan indole, and its high substrate specificity limits the potential of using MibH as a general peptide chlorinase. Other examples include enzymes with unknown function and enzymology, such as the putative O -methyltr ansfer ase r esponsible for N-terminal dimethylation in cacaoidin, or enzymes with an unclear r ecognition motif, suc h as the Fe(II)-2-k etoglutarate-de pendent enzyme PoyI responsible for hydr oxylation in pol ytheonamides. Further genome mining and engineering efforts are required to discover homologues of those enzymes that are better suited for combinatorial applications on a broader series of substrates . Moreo ver, the development of new strategies , e .g. the leader-peptide-free strategy as used for the cyclodehydratase LynD, will further boost the field of lanthipeptide engineering. The engineering studies from different research groups could also be combined to accelerate the de v elopment of molecular engineering of ne w-to-natur e bioactiv e peptides with desir ed structur al pr operties and biological functions, and Fig. 7 depicts this blueprint. Considering the intensive ongoing efforts in RiPPs engineering, it can be expected that many new-to-nature modified peptides will be produced in the near future , pro viding a rich source for new putative antibiotics to be used in human ther a pies. Importantl y, these studies should also be follo w ed b y strong efforts in determining their stability , toxicity , pharmacodynamics , pharmacokinetics , and administration in mammals.

Conflicts of interest statement. None declared.
Funding Y. F. was financially supported by the Chinese Scholarship Council (CSC, Project No. 201806200107). Y. X. was financially supported by the Chinese Scholarship Council (CSC, Project No. 202006210043). F. R. was financially supported by a NWO-ALW OP-415 grant.